Hepatocellular Carcinoma (HCC) is the most common liver malignancy and one of the most major cancer types worldwide. The molecular genetics and signaling pathways involved in HCC pathogenesis remain poorly understood. Hedgehog signaling has been implicated to play important roles in multiple tumor types, including upper gastrointestinal tumors, for example, gastric cancers and pancreatic cancers. Hedgehog signaling has not been studied in HCC. In our preliminary studies, we found that signaling molecules in hedgehog pathways, including, shh, ihh, ptch, smo, Gli1 and hip1, are expressed in both HCC cell lines and tissues. More over, we found that cyclopamine, an inhibitor for hedgehog signaling pathway, is able to inhibit the growth of liver cancer cell lines, including HepG2 and SK-Hep1 cells. In addition, we found that Bmi-1 is highly expressed in HCC compared with adjacent non-tumor liver tissues. A Recent report showed that Bmi-1 can be induced by hedgehog signaling. In this application, we hypothesize that hedgehog signaling is important for development and progression of HCC. Inhibition of hedgehog signaling will suppress HCC tumor growth. We further hypothesize that there are specific gene expression patterns associated with the activation of hedgehog in HCC, and that Bmi-1 is one of the downstream targets of hedgehog signaling. To test these hypotheses, we propose to study the following aims: Aim 1: To characterize the protein expression of hedgehog pathway signaling molecules using tissue microarrays; Aim 2: To study the growth inhibition of hedgehog antagonists in a panel of 14 HCC cells; and Aim 3: To characterize the downstream targets of hedgehog signaling in HCC. In summary, in this exploratory R21 application, we will characterize hedgehog signaling in HCC. These studies may provide crucial insights into HCC pathogenesis that we anticipate could provide a rational target for future therapy for this devastating disease. Identification of novel targets of hedgehog signaling will provide useful information to further characterize hedgehog pathway in HCC.